A system for analyzing the event timing profile of single cells by using a model of neurite maturation in PC12D cells

Nerve growth factor (NGF)-induced neurite maturation in PC12D cells involves neuritogenesis and neurite outgrowth. Actions of compounds affecting the neurite maturation are sometimes invisible behind the individually variable events in nature even in the clonal population. In this study, we designed a time-lapse imaging system to determine the timing of each event in individual PC12D cells. To examine the system, we analyzed the effect of staurosporine on the neurite maturation in PC12D cells. By using the system, we obtained four event timing data sets (stimulation by NGF with and without staurosporine at the concentrations of 0.01, 0.1, and 1 microM). A permutation test of these data sets revealed that staurosporine caused an early induction of neurite outgrowth during neurite maturation in PC12D cells. These results suggest that the time-lapse imaging system to determine the timing of each event in individual cells can provide a novel insight into features of a cell mass by single-cell analysis and is expected to be utilized for profiling of compounds that can serve as candidate drugs.     

Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins

Previously we found that some cyclopentenone prostaglandin derivatives promoted neurite outgrowth from PC12 cells and dorsal root ganglia explants in the presence of nerve growth factor; and so we referred to them as neurite outgrowth-promoting prostaglandins (NEPPs). In this study, NEPPs protected HT22 cells against oxidative glutamate toxicity. NEPP6, one of the most effective promoters of neurite outgrowth in PC12 cells, protected the cells most potently among NEPPs 1--10. Several derivatives, NEPPs 11--19, were newly synthesized based on the chemical structure of NEPP6. NEPP11 had a more potent neuroprotective effect than NEPP6. NEPP11 also prevented the death of cortical neurons induced by various stimuli and reduced ischemic brain damage in mice. Biotinylated compounds of NEPPs were synthesized to investigate their cellular accumulation. NEPP6-biotin protected the cells and emitted potent signals from the cells. In contrast, biotinylated non-neuroprotective derivatives emitted much weaker signals. These results suggest that NEPPs are novel types of neurotrophic compounds characterized by their dual biological activities of promoting neurite outgrowth and preventing neuronal death and that their accumulation in the cells is closely associated with their neuroprotective actions.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Artepillin C Derived from Propolis Induces Neurite Outgrowth in PC12m3 Cells via ERK and p38 MAPK Pathways

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 μM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.     

Melia toosendan regulates PC12 Cell differentiation via the activation of protein kinase A and extracellular signal-regulated kinases

With a history of several thousand years, traditional Chinese medicine has been well documented to be effective in the treatment of various disorders. We have investigated the activities of potential neuroactive compounds in traditional Chinese medicine such as Melia toosendan using an in vitro model system, rat pheochromocytoma PC12 cells. We report here that treatment of PC12 cells with a crude extract of the fruits of M. toosendan reduces cell growth in a dose-dependent manner without detectable cytotoxicity. Upon treatment with M. toosendan, PC12 cells exhibit robust neurite outgrowth, to a greater extent than that observed with nerve growth factor. Results obtained with specific kinase inhibitors and protein kinase A-deficient PC12 cells indicate that the actions of M. toosendan are mediated by the activation of protein kinase A and extracellular signal-regulated kinases.     

Novel NGF-potentiating diterpenoids from a Brazilian medicinal plant,Ptychopetalum olacoides

From the MeOH extract of Ptychopetalum olacoides, which is used in Brazilian folk medicine for the treatment of chronic degenerative conditions of the nervous system, four novel clerodane-type diterpenoids named 6α,7α-dihydroxyannonene (1), 7α,20-dihydroxyannonene (2), 7α-hydroxysolidagolactone I (3), and ptycho-6α,7α-diol (4) were isolated by bioassay-directed fractionation using NGF-differentiated PC12 cells. The structures of 1–4 were established by extensive NMR spectroscopic analyses and chemical conversion. Compounds 1 and 2 significantly enhanced NGF-mediated neurite outgrowth in PC12 cells at concentrations ranging from 0.1 to 50.0 μM for 1 and 0.1 to 30.0 μM for 2, whereas 3 and 4 had no morphological effect on NGF-mediated PC12 cells in the same concentration range. The structure–activity relationship of these compounds is also discussed.     

Phosphorylation of the zebrafish M6Ab at serine 263 contributes to filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos

Background

Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.

Methodology/Principal Findings

We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth.

Conclusions/Significance

Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.     

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.     

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. These authors contributed equally     

Immunosuppressant FK506 induces neurite outgrowth in PC12 mutant cells with impaired NGF-promoted neuritogenesis via a novel MAP kinase signaling pathway

We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Induction of Neurite Outgrowth in PC12 Cells by γ-Lactam-Related Compounds via Ras-MAP Kinase Signaling Pathway Independent Mechanism

Rat pheochromocytoma cells, PC12 cells, undergo differentiation in response to nerve growth factor (NGF). Although the Ras-MAP kinase signaling pathway has been shown to play a central role in the response to NGF, the precise mechanism which induces differentiation remains unclarified. Recently, several γ-lactam-related microbial products were identified to induce neurite outgrowth in neuroblastoma cells. Therefore, we synthesized a series of γ-lactam-related compounds and tested for their ability to induce neurite outgrowth in PC12 cells. We found that two compounds, MT-19 and MT-20, induced neurite outgrowth at concentrations as low as 1 μg/ml. MT-19 and MT-20 have ann-hexadecyl group and ann-dodecyl group, respectively, at the position N-1 of the γ-lactam ring, and the modification of this group leads to partial or complete loss of activity. In addition, the modification of the methyl and hydroxyl group at C-5 leads to complete loss of activity, indicating a strict structure–activity relationship. Interestingly, MT-19 and MT-20 induced neurite outgrowth of PC12 cells which lack normal Ras function. Furthermore, these compounds did not induce MAP kinase activation, suggesting that MT-19 and MT-20 do not require the Ras-MAP kinase signaling pathway which is shown to be necessary and sufficient for NGF-induced neurite outgrowth. Consistent with this, none of the early- or late-response genes tested, which includefos, zif268, Nur77, vgf,and transin, was induced. However, the protein level of three neurofilaments was increased after the incubation with these compounds. Since the level of other cytoskeleton proteins including actin and tubulin remained constant, MT-19 and MT-20 specifically affected neurofilament synthesis and/or turnover. Taken together, these findings indicate that MT-19 and MT-20 induce neurite outgrowth by activating the downstream target of MAP kinase or by a novel mechanism which is distinct from the NGF-activated pathway.     

Minocycline Promotes Neurite Outgrowth of PC12 Cells Exposed to Oxygen-Glucose Deprivation and Reoxygenation Through Regulation of MLCP/MLC Signaling Pathways

Minocycline, a semi-synthetic second-generation derivative of tetracycline, has been reported to exert neuroprotective effects both in animal models and in clinic trials of neurological diseases. In the present study, we first investigated the protective effects of minocycline on oxygen-glucose deprivation and reoxygenation-induced impairment of neurite outgrowth and its potential mechanism in the neuronal cell line, PC12 cells. We found that minocycline significantly increased cell viability, promoted neurite outgrowth and enhanced the expression of growth-associated protein-43 (GAP-43) in PC12 cells exposed to oxygen-glucose deprivation/reoxygenation injury. In addition, immunoblots revealed that minocycline reversed the overexpression of phosphorylated myosin light chain (MLC) and the suppression of activated extracellular signal-regulated kinase 1/2 (ERK1/2) caused by oxygen-glucose deprivation/reoxygenation injury. Moreover, the minocycline-induced neurite outgrowth was significantly blocked by Calyculin A (1 nM), an inhibitor of myosin light chain phosphatase (MLCP), but not by an ERK1/2 inhibitor (U0126; 10 μM). These findings suggested that minocycline activated the MLCP/MLC signaling pathway in PC12 cells after oxygen-glucose deprivation/reoxygenation injury, which resulted in the promotion of neurite outgrowth.     

PC12 cells utilize the homophilic binding site of L1 for cell-cell adhesion but L1-alphavbeta3 interaction for neurite outgrowth

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1-Ig2, indicating that L1-L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti-integrin antibodies suggested the involvement of alphavbeta3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade.     

Cyathane diterpenes from Sarcodon cyrneus and evaluation of their activities of neuritegenesis and nerve growth factor production

Two new cyathane diterpenes, cyrneine C (4) and D (5), were isolated from the mushroom Sarcodon cyrneus, along with previously isolated cyrneine A, B and glaucopine C. The structures of the novel diterpenoids were determined by the analysis of spectroscopic data. Effects of the cyrneines and glaucopine C on the NGF gene expression in 1321N1 cells and on neurite outgrowth on PC12 cells were evaluated.     

(-)-3,5-Dicaffeoyl-muco-quinic acid isolated from Aster scaber contributes to the differentiation of PC12 cells: through tyrosine kinase cascade signaling

Aster scaber T. (Asteraceae) has been used in traditional Korean and Chinese medicine to treat bruises, snakebites, headaches, and dizziness. (-)-3,5-Dicaffeoyl-muco-quinic acid (DQ) isolated from A. scaber induced neurite outgrowth in PC12 cells. It has been reported that the activation of the extracellular signal regulated kinase 1/2 (Erk 1/2) and phosphoinositide 3 (PI3) kinase plays a crucial role in the NGF-induced differentiation of PC12 cells. This study showed that the effect of DQ on neurite outgrowth is mediated via the Erk 1/2 and PI3 kinase-dependent pathways like NGF. Furthermore, DQ stimulated the phosphorylation of Trk A. Overall, DQ elicited the differentiation of PC12 cells through Trk A phosphorylation followed by Erk 1/2 and PI3 kinase activation.     

Phosphatidylcholine biosynthesis via CTP:phosphocholine cytidylyltransferase 2 facilitates neurite outgrowth and branching

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.     

Linckosides C-E, three new neuritogenic steroid glycosides from the Okinawan starfish Linckia laevigata

Three new steroid glycosides, linckosides C-E, were isolated from the Okinawan starfish Linckia laevigata. Their structures and partial stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These metabolites are additional members of the linckosides that were previously discovered as a novel class of neuritogenic compounds. Each of them possesses two monosaccharide units at C-3 of a polyhydroxylated steroidal aglycon and at the side chain (C-28 or C-29). Linckosides C and D are the first steroids that possess a hydroxyisopropyl substituent at C-24 of the side chain. These compounds are not only potent inducers of neurite outgrowth on PC12 cells but also significant enhancers of nerve growth factor (NGF) to induce the neurite outgrowth. The structure-activity relationships within the linckosides revealed that the presence of xylopyranose at the side chain was important rather than arabinofuranose, but that the diversity of the side chain carbon skeleton was not.     

Growth Factor-Like Effects Mediated by Muscarinic Receptors in PC12M1 Cells

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.